Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB）...Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB） and triplex forming oligonucleotide （TFO） are currently developed methods to improve the targeting efficiency. This paper summarized the basic principles, design ideas and application in gene targeting efficiency improvement of these two methods, analyzed and com- pared their characteristics, and finally proposed prospects for their future development.展开更多
This study is used to investigate the feasibility of employing the Iodogen method to label triplex-forming oligonucleotide (TFO) targeted to the initiator of the S gene of HBV with 125I. A 17-mer oligonucleotides sequ...This study is used to investigate the feasibility of employing the Iodogen method to label triplex-forming oligonucleotide (TFO) targeted to the initiator of the S gene of HBV with 125I. A 17-mer oligonucleotides sequence was synthesized and grafted at the 5′ terminal with a tyramine group. Radioiodination of the tyramine-TFO with 125I was then performed using the Iodogen method. After TFO was labeled with 125I using the Iodogen method, the label- ing rate, the radiochemical purity, stability and bioactivity were determined, respectively. The results show that the radiolabeling rate and the radiochemical purity were 93% and 99%, respectively; and the radiochemical purity is more than 90% in vitro at -20°C on the 5th day after labeling; and the rate of 125I-tyramine-TFO binding to HepG2.2.15 cells was (37.2 ± 1.4)% and statistically different from the rate of HepG2 (p < 0.5). Hence, it is concluded that the labeling of oligonucleotides conjugated with tyramine using the Iodogen method is successful and is characterized with a high labeling rate, high stability, and a low loss of bioactivity of the labeled agent.展开更多
An alkaline phosphatase-conjugated 27 - basc oligonucleotide probe was developed to detect the gene, bfPA, encoding the bundle-- forming pilus of enteropathogenic Escherichia coli (EPEC). The probe was proven to be hi...An alkaline phosphatase-conjugated 27 - basc oligonucleotide probe was developed to detect the gene, bfPA, encoding the bundle-- forming pilus of enteropathogenic Escherichia coli (EPEC). The probe was proven to be highly specific to EPEC, hybridizing only toEPEC strains, but not to Salmonella spp. Shigella spp., Vibrio spp., and other E. coli isolates. one hundred seventy eight E. colistrains belonging to traditional EPEC sergroups isolated from patients with diarrhoea were tested the probe, the prevalence rate of bfpAgene was 25. 28%. The frequency of the bfpA gene in relation to serogroups varied widely among EPEc The bfPA gene was morecommonly detected in strains of class l serogroups of EPEC(41.0% ) than in class I serogroups of EPEC(11.6% 6 % ). HEP- 2 cell adhesion assay and the fluorescent actin staining (FAS) test are 100% argreement, with all the localized adherence (LA) positive strainsproucing positive FAS activity. The results of bfpA probe hybridization were of 98.9% agreement with those obtained by using HEP- 2 cell adhesion assay and the FAS test. the sensitivity and specifity of the probe versus the FAS test and the HEP- 2 cell assay were95.7% and 100%, respectively. This probe was proven to be useful in identifying EPEC which carries the bfpA gene.展开更多
PDGF (platelet derived growth factor) has been shown to play animportant role in tumorigenesis, tumor growth, atherosclerosis and inflammation and other various pathologic settings. PDGF-B chain gene is 92% homologous...PDGF (platelet derived growth factor) has been shown to play animportant role in tumorigenesis, tumor growth, atherosclerosis and inflammation and other various pathologic settings. PDGF-B chain gene is 92% homologous to v-sis oncogene of the simian sarcoma virus. Thus PDGF-B gene is also called c-sis proto-oncogene. This report provides 3 TFOs (triplex-forming oligonucleotides) to inhibit the expression of c-sis/PDGF-B gene. The results from gel mobility shift analysis, in vitro transcription, DNase I footprinting and protein binding assays demonstrate that the TFOs we designed can form sequence-specific stable triplex with the target, and can effectively suppress the downstream gene transcription and inhibit transcription factors binding. They can be used for preparation of drugs to inhibit tumor growth and for the therapy of atherosclerosis, inflammation, etc.展开更多
基金Supported by Shandong Swine Industry Technology System and Science and Technology Planning Program for Basic Research in Qingdao City(12-1-4-14-jch)
文摘Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB） and triplex forming oligonucleotide （TFO） are currently developed methods to improve the targeting efficiency. This paper summarized the basic principles, design ideas and application in gene targeting efficiency improvement of these two methods, analyzed and com- pared their characteristics, and finally proposed prospects for their future development.
文摘This study is used to investigate the feasibility of employing the Iodogen method to label triplex-forming oligonucleotide (TFO) targeted to the initiator of the S gene of HBV with 125I. A 17-mer oligonucleotides sequence was synthesized and grafted at the 5′ terminal with a tyramine group. Radioiodination of the tyramine-TFO with 125I was then performed using the Iodogen method. After TFO was labeled with 125I using the Iodogen method, the label- ing rate, the radiochemical purity, stability and bioactivity were determined, respectively. The results show that the radiolabeling rate and the radiochemical purity were 93% and 99%, respectively; and the radiochemical purity is more than 90% in vitro at -20°C on the 5th day after labeling; and the rate of 125I-tyramine-TFO binding to HepG2.2.15 cells was (37.2 ± 1.4)% and statistically different from the rate of HepG2 (p < 0.5). Hence, it is concluded that the labeling of oligonucleotides conjugated with tyramine using the Iodogen method is successful and is characterized with a high labeling rate, high stability, and a low loss of bioactivity of the labeled agent.
文摘An alkaline phosphatase-conjugated 27 - basc oligonucleotide probe was developed to detect the gene, bfPA, encoding the bundle-- forming pilus of enteropathogenic Escherichia coli (EPEC). The probe was proven to be highly specific to EPEC, hybridizing only toEPEC strains, but not to Salmonella spp. Shigella spp., Vibrio spp., and other E. coli isolates. one hundred seventy eight E. colistrains belonging to traditional EPEC sergroups isolated from patients with diarrhoea were tested the probe, the prevalence rate of bfpAgene was 25. 28%. The frequency of the bfpA gene in relation to serogroups varied widely among EPEc The bfPA gene was morecommonly detected in strains of class l serogroups of EPEC(41.0% ) than in class I serogroups of EPEC(11.6% 6 % ). HEP- 2 cell adhesion assay and the fluorescent actin staining (FAS) test are 100% argreement, with all the localized adherence (LA) positive strainsproucing positive FAS activity. The results of bfpA probe hybridization were of 98.9% agreement with those obtained by using HEP- 2 cell adhesion assay and the FAS test. the sensitivity and specifity of the probe versus the FAS test and the HEP- 2 cell assay were95.7% and 100%, respectively. This probe was proven to be useful in identifying EPEC which carries the bfpA gene.
基金the State Key Programs Basic Research of China (Grant No. G1998051103) and National High Technology Programs of China (Grant No. 863-102-08-8).
文摘PDGF (platelet derived growth factor) has been shown to play animportant role in tumorigenesis, tumor growth, atherosclerosis and inflammation and other various pathologic settings. PDGF-B chain gene is 92% homologous to v-sis oncogene of the simian sarcoma virus. Thus PDGF-B gene is also called c-sis proto-oncogene. This report provides 3 TFOs (triplex-forming oligonucleotides) to inhibit the expression of c-sis/PDGF-B gene. The results from gel mobility shift analysis, in vitro transcription, DNase I footprinting and protein binding assays demonstrate that the TFOs we designed can form sequence-specific stable triplex with the target, and can effectively suppress the downstream gene transcription and inhibit transcription factors binding. They can be used for preparation of drugs to inhibit tumor growth and for the therapy of atherosclerosis, inflammation, etc.
